Evolution and Biochemistry of Family 4 Glycosyl Hydrolases Glycosyl hydrolase Family 4 (GH4) is exceptional among the 114 families in this enzyme superfamily. Members of GH4 exhibit unusual cofactor requirements for activity, and an essential cysteine residue is present at the active site. Of greatest significance, is the fact that members of GH4 employ a unique catalytic mechanism for cleavage of the glycosidic bond. By phylogenetic analysis, and from available substrate specificities, we have assigned a majority of the enzymes of GH4 to five sub-groups. Our classification revealed an unexpected relationship between substrate specificity and the presence, in each sub-group, of a motif of four amino acids that includes the active-site Cys residue: alpha-glucosidase, CHE(I/V);alpha-galactosidase, CHSV; alpha-glucuronidase, CHGx; 6-phospho-alpha-glucosidase, CDMP;and 6-phospho-beta-glucosidase, CN(V/I)P. The question arises: does the presence of a particular motif sufficiently predict the catalytic function of an unassigned GH4 protein? To test this hypothesis, we have purified and characterized the alpha-glucoside specific GH4 enzyme (PalH) from the phytopathogen, Erwinia rhapontici.The CHEI motif in this protein has been changed by site-directed mutagenesis, and the effects upon substrate specificity have been determined. The change to CHSV caused the loss of all alpha-glucosidase activity, but the mutant protein exhibited none of the anticipated alpha-galactosidase activity. In conclusion, while the Cys-containing motif may be suggestive of enzyme specificity, and phylogenetic placement can greatly increase confidence in that specificity, the annotation and unambiguous assignment of function of a GH4 protein can be made only on the basis of experimental evidence. Enzymology and Pathogenicity of Streptococcus suis 1. Mannonate dehydratase (ManD) is found only in certain bacterial species, where it participates in the dissimilation of glucuronate. ManD catalyzes the dehydration of D-mannonate to yield 2-keto-3-deoxygluconate (2-KDG), the carbon and energy source for growth. Selective inactivation of ManD by drug targeting, is of therapeutic interest in the treatment of human Streptococcus suis infections. In this collaboration we have over-expressed, purified, functionally characterized and determined the crystallographic structure of ManD from S. suis. Importantly, by Fourier transform mass spectrometry (FTMS),we have shown that 2-KDG is formed when the chemically synthesized substrate (D-mannonate) is incubated with ManD. Inductively coupled plasma-mass spectrometry (ICP-MS) revealed the presence of Mn2+ in the purified protein, and in the solution state catalytically active ManD exists as a homodimer of 41-kDa subunits. The crystal structures of S.suis ManD in native form, and in complex with its substrate and Mn2+ ion, have been solved at a resolution of 2.9 Angstroms. The structure of S. suis ManD is that of a modified form of TIM barrel, similar that of other members of xylose isomerase-like superfamily. Structural analyses, and comparative amino acid sequence alignments, provide evidence for the importance of His311 and Tyr325 in ManD activity. The results of site-directed mutagenesis confirmed the functional role(s) of these residues in the dehydration reaction, and a plausible mechanism for the ManD-catalyzed reaction is proposed. 2. Gluconate 5-dehydrogenase (Ga5DH) is an NADP(H)-dependent enzyme that catalyzes a reversible oxido-reduction between D-gluconate and 5-keto- D-gluconate, thereby regulating the flux of this important carbon and energy source in bacteria. However, despite the considerable amount of physiological and biochemical knowledge of G5DH, there is little physical or structural information available for this enzyme. To this end, we have determined the crystal structures of Ga5DH from the pathogenic organism Streptococcus suis serotype 2 in both native and liganded (NADP+/D-gluconate/metal ion) quaternary complex forms at 1.9 and 1.8 Angstroms resolution, respectively. Structural analysis reveals that Ga5DH adopts a protein fold similar to that found in members of the short chain dehydrogenase/reductase (SDR) family, while the enzyme itself represents a previously uncharacterized member of this family. In solution, Ga5DH exists as a tetramer comprised of four identical 26 kDA subunits. The catalytic site of Ga5DH shows considerable architectural similarity to that found in other enzymes of the SDR family, but the S. suis protein contains an additional residue (Arg104) that plays an important role in substrate binding. The quaternary complex structure provides the first crystallographic evidence for the role of a catalytically important serine residue, and also reveals an amino acid tetrad RSYK that differs from the SYK triad found in the majority of SDR enzymes. Inspection of the crystal structures also reveals the important contributions of metal ions in active site formation, and of residues at the C-termini of subunits to tetramer assembly. Ga5DH is a potential target for therapy, and our findings provide insight not only of the catalytic mechanism, but also for structure-based design of inactivating drugs. Enzymology of Clostridium botulinum Sequencing of the genome of Clostridium botulinum strain Hall A revealed a gene (CBO0515), whose putative amino acid sequence was suggestive of the rare enzyme N5-(1-carboxyethyl) ornithine synthase. To test this hypothesis, CBO0515 has been cloned, and the encoded polypeptide was purified and characterized. This unusual gene appears to be confined to proteolytic strains assigned to Group 1 of C. botulinum.